This invention relates generally to methods and devices for selectively depositing fluids on a nonuniform sample surface according to variations in a surface characteristic. More particularly, the invention relates to the use of nozzleless acoustic ejection to deposit droplets of analysis-enhancing fluid on sample surface sites selected according to the surface characteristic at the sites. The invention is especially useful in enhancing the compositional analysis of the biological samples and in the mass spectrometric imaging of tissue surfaces.
Mass spectrometry is a well-established analytical technique in which sample molecules are ionized and the resulting ions are sorted by mass-to-charge ratio. As the requirements for surface analytical techniques have become more exacting, [advances in mass spectrometry have made it possible to obtain in-depth information regarding a wide variety of sample surface types. In the semiconductor industry, for example, secondary ion mass spectrometry has been used to determine the composition of a microscopic region of a wafer surface. In addition, in the biotechnology arena, surface-based mass spectrometry has been used to analyze single nucleotide polymorphisms in microarray formats. See, e.g., U.S. Pat. No. 6,322,970 to Little et al.
Matrix-Assisted Laser Desorption Ionization (MALDI) is an ionization technique often used for mass spectrometric analysis of large and/or labile biomolecules, such as nucleotidic and peptidic oligomers, polymers, and dendrimers, as well as for analysis of nonbiomolecular compounds, such as fullerenes. MALDI is considered a xe2x80x9csoftxe2x80x9d ionizing technique in which both positive and negative ions are produced. The technique involves depositing a small volume of sample fluid containing an analyte on a substrate comprised of a photon-absorbing matrix material selected to enhance desorption performance. See Karas et al. (1988), xe2x80x9cLaser Desorption Ionization of Proteins with Molecular Masses Exceeding 10,000 Daltons,xe2x80x9d Anal. Chem., 60:2299-2301. The matrix material is usually a crystalline organic acid that absorbs electromagnetic radiation near the wavelength of the laser. When co-crystallized with analyte, the matrix material assists in the ionization and desorption of analyte moieties. The sample fluid typically contains a solvent and the analyte. Once the solvent has been evaporated from the substrate, the analyte remains on the substrate at the location where the sample fluid has been deposited. Photons from a laser strike the substrate at the location of the analyte and, as a result, ions and neutral molecules are desorbed from the substrate. MALDI techniques are particularly useful in providing a means for efficiently analyzing a large number of samples. In addition, MALDI is especially useful in the analysis of minute amounts of sample that are provided over a small area of a substrate surface.
Surface Enhanced Laser Desorption Ionization (SELDI) is another example of a surface-based ionization technique that allows for high-throughput mass spectrometry. SELDI uses affinity capture reagents such as antibodies to collect samples from a complex mixture, which allows in situ purification of the analyte, followed by conventional MALDI analysis. Typically, SELDI is used to analyze complex mixtures of proteins and other biomolecules. SELDI employs a chemically reactive surface such as a xe2x80x9cprotein chipxe2x80x9d to interact with analytes, e.g., proteins, in solution. Such surfaces selectively interact with analytes and immobilize them thereon. Thus, analytes can be partially purified on the chip and then quickly analyzed in the mass spectrometer. By providing different reactive moieties at different sites on a substrate surface, throughput may be increased.
Recently, mass spectrometry techniques involving laser desorption have been adapted for cellular analysis. U.S. Pat. No. 5,808,300 to Caprioli, for example, describes a method for imaging biological samples with MALDI mass spectrometry. This method allows users to measure the distribution of a specific element or small molecule within biological specimens such as tissue slices or individual cells. In particular, the method can be used for the specific analysis of peptides in whole cells, e.g., by obtaining signals for peptides and proteins directly from tissues and blots of tissues. In addition, the method has been used to desorb relatively large proteins from tissues and blots of tissues in the molecular weight range beyond about 80 kilodaltons. From such samples, hundreds of peptide and protein peaks can be recorded in the mass spectrum produced from a single laser-ablated site on the sample. When a laser ablates the surface of the sample at multiple sites and the mass spectrum from each site is saved separately, a data array is produced, which contains the relative intensity of any given mass at each site. An image of the sample surface can then be constructed for any given molecular weight, effectively representing a compositional map of the sample surface.
One important issue to successful MALDI profiling and imaging as described above is the application of a mass spectrometry matrix material to the tissue surface at each site of laser ablation. As described in Caprioli, the mass spectrometry matrix material may be applied as a continuous and uniform coating of less than about 50 micrometers in thickness. In order to apply the mass spectrometry matrix material in a controlled manner, carefully metered amounts of sample fluids should be accurately and precisely placed on a sample surface. Acoustic ejection is a technique that is well suited for depositing minute volumes of fluids on a surface because the technique allows for control over droplet volume and thus xe2x80x9cspotxe2x80x9d size on the surface, as well as control over the trajectory of ejected droplets and the precise location of the deposition sites on the surface. See, e.g., U.S. Patent Application Publication No. 20020037579 to Ellson et al. In particular, U.S. patent application Ser. No. 10/087372, entitled xe2x80x9cMethod and System Using Acoustic Ejection for Preparing and Analyzing a Cellular Sample Surface,xe2x80x9d filed Mar. 1, 2002, by inventors Ellson, Mutz, and Caprioli, describes the use of nozzleless acoustic ejection to deposit mass spectrometry matrix material at designated sites on a sample surface to form either a uniform matrix material layer or an array of individual sites. In some instances, different analysis-enhancing fluids may be applied to an analyte to optimize experimental parameters.
As with many types of samples, cellular samples are not typically uniform in composition, and the distribution of materials on the surface of such samples may vary. In addition, the sample surfaces may exhibit inhomogeneous morphologies. Since certain analysis-enhancing fluids are appropriate for use with certain analytes, there is a need to selectively deposit the analysis-enhancing fluid according to the surface characteristics of the cellular sample at that site. Acoustic ejection provides a means for carrying out such fluid deposition with unparalleled accuracy, precision, and efficiency.
Accordingly, one embodiment of the invention relates to a method for selectively depositing an analysis-enhancing fluid on a sample surface. The method involves providing a sample having a surface that exhibits variations in a specific characteristic, which corresponds to its desirability for receiving an analysis-enhancing fluid. Once a site on the sample surface has been selected according to the desired surface characteristic, focused radiation, typically acoustic radiation, is applied in a manner effective to eject a droplet of the analysis-enhancing fluid from a reservoir. As a result, the droplet is deposited on the sample surface at the selected site. In some instances, a plurality of sites is selected, and a droplet of the analysis-enhancing fluid is deposited onto each selected site. The sites may form a single contiguous region on the sample surface or a plurality of noncontiguous regions on the sample surface. Optionally, the sample at the selected sites is analyzed.
Although the invention may be practiced using any sample having a nonuniform surface, cellular samples are particularly well suited. Examples of suitable cellular samples include, for example, tissue samples, cell cultures, single cells, or a plurality of cells immobilized on a substrate surface. In addition, the invention is particularly suited for samples having a substantially planar surface.
The surface characteristic corresponding to desirability for receiving analysis-enhancing fluid is typically a compositional or a morphological characteristic that is optically and/or acoustically detectable. For example, when the sample surface is a tissue sample comprised of distinct fatty and lean regions, it may be desirable to deposit analysis-enhancing fluid on only the fatty region. In such a case, the sites for fluid deposition may be selected according to lipid and/or peptide content at the site. Thus, the analysis-enhancing fluid may be selected according to the type of analysis desired. In some instances, the analysis-enhancing fluid comprises a label moiety, such as one or more selected from the group consisting of a fluorescent moiety, a magnetic moiety, and a radioactive moiety. In addition or in the alternative, the analysis-enhancing fluid may contain a biomolecule, nucleotidic, peptidic, or otherwise. Often, an enzymatic biomolecule is provided to enhance breakdown of the corresponding substrate moiety on or near the sample surface.
In some instances, the sample may exhibit variations in a plurality of surface characteristics, wherein each characteristic corresponds to desirability for receiving a different analysis-enhancing fluid. In such a case, droplets of different analysis-enhancing fluids may be deposited on the sample surface at the corresponding selected sites. In addition, different analysis-enhancing fluids may be deposited on the sample surface at the same site.
The invention is particularly suited for mass spectrometric analysis. In order to carry out a surface-based mass spectrometry technique, mass spectrometry matrix material may be deposited on the sample surface, and energy is then applied to the deposited matrix material to cause release of sample molecules from the sample surface for analysis. Typically, the matrix material is a photoabsorbing matrix material, and a photon source such as a laser is used to ionize the sample.
Thus, the invention is also particularly suited for use with sample surface imaging. In some instances, a sample surface is imaged so as to map the variations in the surface characteristic, and one or more sites are selected on the sample surface using the results of surface imaging. Preferably, surface imaging results in the production of a digital image. Optionally, the imaging contrast of the variations in the surface characteristic is increased before the surface is imaged. This may involve, for example, staining the sample surface such that regions exhibiting the desired surface characteristic are more readily resolved by an imaging means.
In another embodiment, the invention provides a system for selectively depositing an analysis-enhancing fluid on a surface of a sample. The system includes a reservoir containing an analysis-enhancing fluid, an acoustic ejector comprising an acoustic radiation generator for generating acoustic radiation and a focusing means for focusing the acoustic radiation generated, and a means for positioning the acoustic ejector in acoustic coupling relationship to the reservoir. Also provided is a means for selecting at least one site on the sample surface for deposition of analysis-enhancing fluid thereon. Site selection is carried out according to a surface characteristic that corresponds to desirability for receiving the analysis-enhancing fluid. The system also includes a means for positioning the sample such that the selected site or sites are positioned in droplet-receiving relationship to the reservoir. The sample positioning means may be adapted to controllably position the sample such that when a plurality of sites on the sample surface is selected, the selected sites are successively placed in droplet-receiving relationship to the reservoir.
In some instances, the selecting means is comprised of a means for imaging of the sample surface so as to map the variations in the surface characteristic and a means for selecting at least one site on the sample surface using the results produced by the imaging means. The imaging means is typically adapted to produce a digital image and may, for example, employ a scanner or a camera. In other instances, an image of the sample surface that maps the variations in the surface characteristic is produced without an imaging means. For example, a low-resolution map of the compositional characteristics of the sample could be formed based on performing mass spectrometry of spots from a sparse grid of analysis-enhancing fluid deposited on the sample. In either case, the selecting means may select sites for analysis-enhancing fluid deposition when the surface characteristic is above a threshold level, below a threshold level and/or when the surface characteristic is within a predetermined range.
The inventive system may further include a means for analyzing the composition of the sample at the selected site. The analyzing means may include a mass spectrometer, an optical detector, a radiation detector, and/or a magnetic detector. A means for applying energy to the sample surface may be provided as well to effect release and ionization of sample molecules from the sample surface for analysis. For example, the energy applying means may include a laser and/or other means for bombarding the surface with photons.
In a further embodiment, the system may include a plurality of reservoirs, each containing a different analysis-enhancing fluid. In such a case, the site selecting means may select sites according to a surface characteristic that corresponds to desirability for receiving at least one of the analysis-enhancing fluids. In addition, the sample positioning means positions the sample such that the selected sites thereon are placed successively in droplet-receiving relationship to the reservoir containing the analysis-enhancing fluid that corresponds to the surface characteristic at the selected sites.
In still another embodiment, the selecting means represents an optional component of system, and the sample positioning means positions the sample such that at least one site on the sample surface exhibiting a surface characteristic that corresponds to desirability for receiving the analysis-enhancing fluid is placed in droplet-receiving relationship to the reservoir. This and other embodiments may be adapted to controllably position the sample such that when a plurality of sites on the sample surface exhibits the desired surface characteristic, the selected sites are successively placed in droplet-receiving relationship to the reservoir. In addition, the sample positioning means may include digital information that relates the location or locations for analysis-enhancing fluid deposition.